It has long been recognized that it is, in many cases, desirable to provide a targeted site with a property or function to the exclusion of the environment which surrounds this site. Early examples of such specific targeting include the design of immunotoxins where, in principle, a toxic moiety could be delivered selectively and specifically to a cellular target where the toxicity was desired without negative side effects on the surrounding tissue. It may also be desirable, as further described below, to provide an enzymatic activity at a particular location to the exclusion of the surroundings. Typically, attempts have been made to accomplish this by using a single ligand to carry the active principle to the desired location, relying on the affinity of the ligand for the targeted site to provide selectivity.
However, in many instances, this approach does not confer sufficient selectivity. For example, it has been established that the pharmacokinetics of antibody binding to tumor cells is slow (24-48 hours for maximal specificity). During this time, a toxic effector coupled to the antibody would be expected to have deleterious effects on the remainder of the system. Coupling the antibody against a tumor cell surface antigen to avidin, has been suggested as a way to avoid systemic exposure to toxic effectors. Administering a biotin conjugated toxin or radioligand after the antibody is maximally localized results in clearing of the small molecule from the body in less than one hour, thereby greatly reducing systemic exposure (Sung and van Osdol, J. Nucl. Med. (1995) 36(5):867-76).
Similarly, antibody-enzyme conjugates have been used to activate small molecule prodrugs administered after the antibody conjugate has maximally localized (Melton and Sherwood, J. Natl. Cancer Inst. (1996) 88(3-4):153-65). This strategy is commercialized by Seattle Genetics, Inc. Enzymatic activation of a prodrug extracellularly is particularly desirable for treating cancer because it creates a bystander effect for killing tumor cells in the vicinity. Thus, the targeting antigen can be as simple as histones, present in large amounts in necrotic cell debris within most solid tumors.
Still another variation of the basic antibody-effector conjugate idea is exemplified by the TAP (tumor activated prodrug) technology commercialized by Immunogen, in which an antibody conjugated to a toxin, e.g., maytansine, is internalized preferentially by cells expressing the relevant antigen for that antibody, with toxin released intracellularly (Liu and Chari, Exp. Opi. Invest. Drugs (1997) 6:169-172). A drawback of the TAP approach is that it lacks a bystander effect, thus selecting for mutants that have lost the targeting antigen.
All of these existing techniques suffer from the intrinsic limits on specificity of the targeting antibody. In this aspect, target cells are typically a small fraction of all cells in the body, often in the neighborhood of 1/100,000 or less. Accordingly, even if the antibody has very high specificity, with affinity for target antigen being 100,000 times higher than for any other antigen in the body, the fraction of the conjugated effector that is distributed to non-target cells is still 50%. In practice, antibody specificities are not usually this high, and the background binding is correspondingly higher. Thus, in this context, the use of a single targeting ligand is generally less than satisfactory.
The goal of the present invention is to improve the specificity of targeting in general by requiring multiple independent binding events before an effector function is created. This approach is not limited to delivering effector functions to the surface of cells in vivo, but can also be used to deliver effector functions to intracellular targets, and to cellular or non-cellular targets in vitro.
The invention also provides specific methods and kits for binary or polynary targeting to create an enzymatic activity, of particular utility for activating prodrugs at cell surfaces. The invention further provides methods and kits for assaying an analyte using binary or polynary targeting.
A natural example for binary reconstitution of an effector is the gramicidin toxin, which creates a pore through the cell membrane by end to end dimerization of an ion channel that spans half the width of the membrane. In nature, this particular toxin can flip in the plane of the membrane so it is not necessary to target one monomer to the inside and one to the outside of the cell in order to create the effector function. Although these precedents suggest that the present invention may be embodied in a naturally existing construct, it is normally necessary to alter and/or combine several moieties to make a targeting component with all the requisite properties. The present invention excludes such naturally occurring phenomena.